Taxanes covalently bounded to hyaluronic acid or hyaluronic acid derivatives

ABSTRACT

The present invention relates to water-soluble taxanes covalently bounded tohyaluronic acid or hyaluronic acid derivatives, and in particular to paclitaxel and docetaxel, useful for the preparation of pharmaceutical compositions to be used in the field of oncology, in the treatment of autoimmune disorders and of restenosis. The invention also relates to the process for preparing taxanes covalently bounded to hyaluronic acid or hyaluronic acid derivatives by direct synthesis between molecules of hyaluronic acid and of taxane or by indirect synthesis by the introduction of a spacer between the hyaluronic acid or hyaluronic acid derivative and the taxane.

FIELD OF THE INVENTION

The present invention relates to taxanes, in particular paclitaxel anddocetaxel, covalently bounded to hyaluronic acid or hyaluronic acidderivatives, to the process for their preparation and to their use inthe field of oncology, in the treatment of autoimmune disorders andrestenosis.

STATE OF THE ART

Taxanes, and in particular paclitaxel and docetaxel, currently marketedunder the trade names Taxol® and Taxotere®, are anticancer agents(Huizing M. T. et al., Cancer Inv., 1995, 13: 381-404) that exert theirantiproliferative effect by acting on the organisation of themicrotubules in the cellular cytoskeletal system. Indeed, by inhibitingthe depolarisation of said microtubules, they prevent their normaldynamic reorganisation that occurs during mitotic cell division(Manfredi J. J. et al., J. Cell Biol., 1982, 94: 688-696).

The main therapeutic indications for paclitaxel are:

therapy for advanced breast cancer;

therapy for Kaposi's sarcoma;

therapy for carcinoma of the lung (not microcytoma)

carcinoma of the ovary, resistant to standard chemotherapy treatment.

Moreover, said chemotherapy is also used to treat carcinoma of thebladder, prostate and endometrium.

Given that paclitaxel is insoluble in water, it is mixed with Cremophor®EL (castor oil)—ethyl alcohol in a ratio of 1:1, in the pharmaceuticalcompositions currently used in cancer chemotherapy (Pfeifer R. W. etal., Am. J. Hosp. Pharm., 1993, 50:2520-2521). This formulation isusually used for continuous intravenous infusion (for between 3 and 24hours) at a dosage of 135-175 mg/m².

The presence of Cremophor® EL in the above said formulation is the maincause of the adverse reactions that normally occur during administrationof paclitaxel, ranging from simple attacks of urticaria to dyspnea andbronchospasm, and even anaphylactic shock (Weiss, R. B. et al., J. Clin.Oncol., 1990, 8: 1263-1268).

For this reason, any patient who is going to receive treatment with apharmaceutical composition of paclitaxel-Cremophor® EL must first followa premedication protocol, with the administration of dexamethasone,possibly associated with an antihistamine.

In spite of these precautions, up to 40% of the patients who receiveintravenous infusion of paclitaxel still experience more or less severeadverse reactions. It can therefore be said that the formulation ofTaxol® currently in clinical use, and the methods used for administeringit, constitute a limitation to its efficacy. This is the reason whyresearch is now being directed towards the synthesis of newpharmaceutical formulations and/or towards new chemical formulations ofthe above anticancer drug, that are water-soluble.

For instance, researchers have attempted to encapsulate paclitaxel inliposomes, nanocapsules and microspheres constituted by a polymer wallformed by biodegradable co-polymers, such as polylactic acid, andnon-biodegradable co-polymers, such as ethylene-vinyl-acetate.

Moreover, microspheres have been prepared that are loaded withpaclitaxel formed by a biodegradable polymer, such as polyphosphoester,to create a system for the prolonged release of drug at the treatmentsite in the therapy for lung carcinoma (Nuijen, B. et al.,Investigational New Drugs, 2001, 19: 143-153). There have also beenattempts to prepare micelles of said anticancer drug by precipitatingpaclitaxel in an organic solvent with phosphatidylcholine/bile salts(Nuijen, B. et al., Investigational New Drugs, 2001, 19: 143-153).

However, these new systems for the encapsulation of paclitaxel may provetroublesome with regard to stability, production and reproducibility.

Moreover, various attempts have been made to dissolve the drug withcyclodextrine, but the new formulations did not give the desired results(Nuijen, B. et al., Investigational New Drugs, 2001, 19: 143-153).

Chemical research into new formulations of paclitaxel that render thedrug more water-soluble while maintaining its efficacy as an anticanceragent, has led to the synthesis of new analogues modified at the C2^(I)and C7 position. (US patent application No. 2001/0018531) as well as tothe preparation of new prodrugs.

Prodrugs are therapeutically inert drug derivatives that are activatedby being introduced into a body. There, after spontaneous hydrolysisand/or enzymatic degradation processes, the active principle isreleased.

In view of this, and for the above said reasons, many attempts have beenmade to synthesise new prodrugs which have led, for instance, to thepreparation of drugs such as acetyl-paclitaxel (Mellado, W. et al.,Biochem. Blophys. Res. Commun., 1984, 124(2): 329-336), or to thesynthesis of new esters of said drug with succinic, glutaric andsulphonic acid on the carbon in position C2^(I). These esters, however,proved to be unstable in aqueous environment.

Moreover, some derivatives with a phosphonoxyphenylpropionate estergroup at the C2^(I) or C7 position have been synthesised, such aspaclitaxel-2^(I)-carbonate, and a series of new amino acid esters ofpaclitaxel and derivatives thereof, with a glutaryl group at positionC2^(I).

Glutaryl-paclitaxel asparagine and glutaryl-paclitaxel glutamine haveproved to be the two most highly water-soluble products obtained by thetype of synthesis described above, but they are less efficacious thanpaclitaxel per se (Nuijen, B. et al., Investigational New Drugs, 2001,19: 143-153).

It is also known that paclitaxel has been esterified withpoly-L-glutamic acid to form a new water-soluble derivative of saidchemotherapy drug, with a significantly higher plasma half-life thannon-conjugated paclitaxel (Li C. et al., Cancer Research, 1998, 58(11):2404-2409).

Paclitaxel has also been derivatised with PEG (polyethylene glycol) byesterifying the chemotherapy drug at position C2^(I); however, the newmolecule has proved to be highly water-soluble but not very stable.

Lastly, a new delivery system for the drug has recently been developed,by the conjugation of paclitaxel with human serum albumin (HSA). Thepaclitaxel-HSA conjugate has proved to be very water-soluble and capableof carrying up to 30 molecules of chemotherapy drug. However,experiments performed in vitro have shown it to be less efficaciousagainst cancer than paclitaxel per se (Nuijen, B. et al.,Investigational New Drugs, 2001, 19:143-153).

Recently, researchers have synthesized a new delivery system forpaclitaxel esterified with previously modified hyaluronic acid(hereinafter referred to as “HA”), that is HA reacted with molecules ofhydrazide bound to the carboxyl group of HA by an amide bond (Luo Y. etal., Biomacromolecules 2000, 1 (2): 208-218; U.S. Pat. No. 5,874,417).This new delivery system for paclitaxel enables the drug to go directlyto the membrane surface of the target cancer cell, characterized byoverexpression of the receptor for HA, CD44. Consequently, thepaclitaxel bounded to HA functionalized with a hydrazide proves to beable to bind specifically to the CD44 of the cancer cell, and it is thusenabled (thanks to a process of endocytosis) to enter the cell cytoplasmwhere it can be enzymatically released and activated, triggering itsinhibition of the depolarization of tubuline and therefore of cellulardivision. This mechanism of selective transport of the drug is called“cell targeting”.

Moreover, it is known that HA can be used as a vehicle for anticancerdrugs in pharmaceutical compositions wherein HA is associated with (andnot covalently bound to) chemotherapy drugs, such as paclitaxel, toincrease their therapeutic. efficacy thanks to the targeting phenomenondescribed above (International Patent Application No. WO 00/41730) andto enable the doses commonly specified in normal chemotherapy protocolsto be lowered (International Patent Application No. WO 99/02151).

Lastly, low-molecular-weight HA and/or the lipid derivatives thereof areknown to be used to prepare liposomes used for the delivery of drugs,including anticancer drugs such as paclitaxel (International PatentApplication No. WO 01/39815).

In view of what said above, it is still felt the need of novel taxanesderivatives, which are stable and soluble in water, and therapeuticallyefficacious at least so as the not-modified taxanes are.

SUMMARY OF THE INVENTION

The Applicant has now found that covalently bounding taxanes to HA or HAderivatives, optionally by means of a spacer, stable and water-solubleproducts are obtained, useful for the preparation of pharmaceuticalcompositions for the treatment of tumours, autoimmune disorders andrestenosis.

It is therefor subject of the invention a taxane covalently bounded toHA or to a HA derivative, wherein the covalent bond is formed betweenhydroxyl groups of the taxane and carboxyl groups or hydroxyl groups ofHA or of HA derivatives, or amino groups of deacetylated HA, optionallyby means of a spacer linking the taxane to HA or HA derivative, with theproviso that the said spacer is different from a hydrazide.

The present invention further relates to the processes for thepreparation of taxanes covalently bounded to HA or HA derivatives.

Further subject of the invention are the pharmaceutical compositionscomprising as the active substance at least a taxane covalently boundedto HA or HA derivatives, and their use in the treatment of tumours,autoimmune disorders and restenosis.

The present taxanes covalently bounded to HA or HA derivatives have manyadvantages, which may be summarised as follows:

1) they are instantly soluble in the bloodstream;

2) they do not need to be mixed with Cremophor® El for the preparationof formulations, thus overcoming the aforesaid problems concerninghypersensitivity and anaphylaxis;

3) thanks to the enzymatic action of enzymes such as the esterasescommonly present in plasma, the taxanes are instantly released by theirvehicle HA or HA derivative from the present compositions into theblood, where they can freely perform their anticancer activity;

4) they enable a new drug to be obtained which, in the case of certaintypes of cancer, may exert surprising chemotherapy activity that issignificantly greater than that obtained when a non-conjugated taxane isadministered, when same doses of drug are considered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the percentage of survival after implant of neoplasticcells as described in Example 1 for controls (black histogram), and formice who received paclitaxel (grey histogram), and paclitaxel covalentlybounded to HA ester with 16% of esterification (white histogram)prepared as in Example 7.

FIG. 2 shows the pharmacological power expressed as IC50 and resultingfrom experiments in Example 2, of the paclitaxel covalently bounded toester derivatives of HA having 16% esterification (grey histogram), 22%of esterification. (black histogram) and 6.8% of esterification (whitehistogram) for four cell lines of breast cancer, vs. the referenceproduct paclitaxel.

FIG. 3 shows the percentage of survival after implantation of neoplasticcells as described in Example 3, in control mice (broken line) and inmice who received ACP® gel (continuous line).

FIG. 4 shows the percentage of paclitaxel covalently bounded to HA esterprepared as described in Example 7, released in human plasma asdescribed in test of Example 13, vs. time.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes compounds belonging to the taxanefamily, preferably paclitaxel and docetaxel hereinafter represented bythe formulae (I) and (II) respectively, covalently bounded to HA or HAderivatives, preferably by means of a spacer as an interface between thetaxane component and the HA or HA derivative, being covalently bound toboth molecules.

HA is a hetero-polysaccharide composed of alternate residues ofD-glucuronic acid and N-acetyl-D-glucosamine, having the followingrepeating unit:

HA is a linear-chain polymer with a molecular weight which may varybetween. 50,000 and 13×10⁶ Da, according to its source and the methodused to obtain it. It is present in nature in the. pericellular gels, inthe fundamental substance of connective tissue in vertebrate organisms(of which it is one of the main components), in the synovial fluid ofjoints, in the vitreous humor and in the umbilical cord. HA plays animportant role in the biological organism, as a mechanical support forthe cells of many tissues such as the skin, the tendons, the muscles andthe cartilage. It is the main component of the extracellular matrix, butit has other functions, such as the hydration of tissues, lubrication,and cell migration and differentiation.

The HA used in the present invention may be extracted from any source,for example from cocks' combs, or it may be obtained by the fermentationroute, or by technological means, and it may have a molecular weight ofbetween 400 and 3×10⁶ Da, in particular between 400 and 1×10⁶ Da, andpreferably, between 400 and 230,000 Da.

The HA derivatives according to the present invention are preferablyselected from the group consisting of the following HA derivatives:

HA salified with organic and/or inorganic bases;

Hyaff®: HA esters with alcohols of the aliphatic, araliphatic;cycloaliphatic, aromatic, cyclic and heterocyclic series, with anesterification degree that may vary according to the type and length ofthe alcohol used, and is in any case never over 50% esterification, andpreferably between 0.1 and 20% since the final polymer that is obtainedmust always be water-soluble, while the remaining percentage ofnon-esterified HA may be salified with organic and/or inorganic bases,disclosed in U.S. Pat. No. 4,851,521 incorporated herewith by reference;

Hyadd™: amides of HA with amines of the aliphatic, araliphatic,cycloaliphatic, aromatic, cyclic and heterocyclic series, with apercentage of amidation of between 0.1 and 10%, since the final polymermust always be water-soluble, while the remaining percentage of HA thatis not amidated can be salified with organic and/or inorganic bases,disclosed in European patent application No. 1095064 incorporatedherewith by reference;

O-sulphated HA derivatives to the 4^(th) degree of sulphation, disclosedin U.S. Pat. No. 6,027,741 incorporated herewith by reference;

ACP®: inner esters of HA with a percentage of esterification of no morethan 15%, as the polymer must always be water-soluble, preferablybetween 0.05 and 10% of esterification, while the remaining percentageof unesterified HA can be salified with organic and/or inorganic bases,disclosed in European patent No. 0341745 B1 incorporated herewith byreference;

deacetylates of HA: these derive from the deacetylation of theN-acetyl-glucosamine unit with a percentage of deacetylation preferablybetween 0.1 and 30% while all the carboxylic groups of HA can besalified with organic and/or inorganic bases, as illustrated in thefollowing structure (A):

Deacetylates of HA are disclosed in International Patent Application No.WO 02/18450 we incorporate herewith by reference;

Hyoxx™: percarboxylated HA derivatives obtained by oxidation of theprimary hydroxyl of the N-acetyl-glucosamine unit with a degree ofpercarboxylation of between 1 and 100%, preferably between 25 and 75%.All the carboxylic groups of HA can be salified with organic and/orinorganic bases as illustrated in the following structure (B):

Percarboxylated HA derivatives are disclosed in US Patent ApplicationNo. US2003181689.

Moreover, the present compounds wherein a taxane, and in particularpaclitaxel, is covalently bounded to an HA ester, may be obtained bystarting from molecules of chemically unmodified HA and, only aftersynthesis with the chemotherapy drug, modifying the HA by esterifying itwith all the alcohols listed above for the Hyaff® products, or byforming inner esters as in the case of ACP® (see Example 8).

The previously listed HA derivatives, that are particularly important inthe process of synthesis of the prodrug HA-taxane, and in particular ofthe prodrug HA-paclitaxel, are the deacetylated and sulphatedderivatives because at the same percentages of paclitaxel bound topreviously unmodified hyaluronic acid, they render the final productmore soluble in the bloodstream.

It is known that, by means of the CD44 membrane receptor, HA modulatesmany different processes relative to cell physiology and biology such asthe proliferation, differentiation and locomotion of cancer cells andother cells.

Scientific literature has recently demonstrated the efficacy of HAagainst cancer, when HA is injected as such directly into the cancergrowth. It has proved to be able to determine the complete regression of30% of tumours (HerreraGayol, A. et al., Experimental and MolecularPathology, 2002, 72: 179-185).

It is also known that HA can be associated with any chemotherapy drug toprepare many different pharmaceutical compositions, as it is able to actas a second antineoplastic agent that synergically enhances theanticancer action of the drug associated with it (International PatentApplication No. WO 01/47561); alternatively, HA is claimed as ananticancer drug to be administered on its own in various clinicalprotocols for the reduction/regression of the cancer growth(International Patent Application No. WO 97/40841).

The present taxane covalently bounded to HA or HA derivatives, as abovesaid, differs from all the formulations of taxanes, in particular thecovalent bound of paclitaxel with HA or HA derivatives, optionally bymeans of a spacer, renders the paclitaxel soluble in water, withoutdiminishing its pharmacological efficacy, Indeed, the in vivo experimentdescribed in Example 1 clearly demonstrates the same anticancer efficacyof the present conjugated paclitaxel and non-conjugated paclitaxel, whensame doses are administered.

Moreover, HA-paclitaxel can present unexpected pharmacologicalproperties that are different from those of the non-conjugatedpaclitaxel, especially in the case of certain types of tumour.

Indeed, Example 2 clearly demonstrates that the present ester derivativeof HA bounded to paclitaxel has a new antineoplastic pharmacologicalactivity: in the model of in vitro cytotoxicity described hereafter, thepresent HA-paclitaxel shows surprising anticancer activity that is farsuperior to that exerted by non-conjugated paclitaxel alone.

This new antineoplastic property means that the present taxanes, inparticular the paclitaxel, conjugated to HA or HA derivatives, can beused for the preparation of pharmaceutical compositions useful as achemotherapy drug, not only for the treatment of all the forms of tumourfor which Taxol® is administered, but also for other forms of tumour notnormally treated with Taxol®, such as cancer of the stomach and liver,cancer of the colon, melanoma and leukaemia. Moreover, it can be used insystemic autoimmune disorders such as rheumatoid arthritis, systemiclupus erythematosus, autoimmune glomerulonephritis and, lastly,Hashimoto's thyroiditis.

The use of the present products in a new pharmacological therapy for theabove said pathologies is possible because the new HA-paclitaxelcompound reduces the systemic toxicity of Taxol®thus increasing thetherapeutic efficacy of the drug itself, since it is:

water-soluble;

not associated with Cremophor® EL and is therefore free from the toxiceffects that this produces;

equally efficacious at doses decidedly lower than (or equal to) thosenormally used in clinical protocols.

Also known is the use of paclitaxel as a drug to be used to inhibit theprocess of restenosis that generally follows angioplasties (prevalentlyarterial), coronary bypass and organ transplants.

The present taxanes, and in particular the paclitaxel, covalentlybounded to HA or HA derivatives can also be used for the prevention ofrestenosis or they can be used to form an inner coating for stents anddevices implanted after the above-listed vascular operations, as it hasproved possible to bind it chemically to the surface of said stents orto adsorb it easily to them.

In either case, the residence time of the present products on thesurface of the stent, and consequently its gradual release into thebloodstream, is greater than that of non-conjugated paclitaxel becausethe chemical-physical characteristics of HA favour a progressive, slowbut continuous release of Taxol® from the surface of the device.

The pharmaceutical compositions comprising the present taxanescovalently bounded to HA or HA derivatives can be administeredsystemically (by the intravenous or arterial, intramuscular,intraperitoneal, subcutaneous or oral routes), it can be used fortopical application (by transdermal absorption), or it can beadministered directly into the cancer site by means of injection. HA ora derivative thereof covalently bound to paclitaxel, can also act as ananticancer drug per se.

In the following Example 3, the Applicant demonstrates how treatment ofexperimentally-induced tumour growths in nude mice with the cross-linkedderivative of HA, ACP®, determines a significant regression of thetumour compared to the non-treated controls.

The Applicant therefore describes for the first time a new role for HAand the derivatives thereof that constitute the present productstaxane-HA or taxane-HA derivative, as antineoplastic agents and theirrelative uses in the field of oncology. The present taxanes covalentlybounded to HA or HA derivatives can, moreover, be associated withvarious biologically and pharmacologically active molecules such as, forexample, steroids, hormones, proteins, trophic factors, vitamins,non-steroid anti-inflammatory drugs, chemotherapy drugs,calcium-antagonists, antibiotics, antiviral agents, interleukins andcytokines such as Interferon.

In this way, it is possible to obtain many different associations of theabove said drugs and relative different pharmaceutical compositionscomprising the taxanes of the invention.

The present Invention also relates to the process for preparing thepresent taxanes, in particular paclitaxel, covalently bounded to HA orHA derivatives; the present products may be achieved by the followingprocesses:

1) by an indirect synthesis that involves the introduction of a spacerbetween the taxane and HA or HA derivative, or

2) by a direct synthesis between the taxane and HA or HA derivative.

The functional groups of HA or HA derivatives that can react with thetaxane directly or indirectly by means of a spacer, are the following:

1) hydroxyl groups;

2) carboxyl groups;

3) amino groups of deacetylated HA.

The spacer may be for example selected from the group consisting of analiphatic or araliphatic chain, linear or branched, substituted by oneor more groups selected from hydroxyl, carboxyl or carbonyl groups,epoxides, acyl chlorides, mercaptans, nitryls, halogens, anhydrides,isocyanates and isothiocyahates, and amino groups.

Amongst the possible spacers, the bromides of carboxylic acids havingfrom 2 to 18 carbon atoms are preferable, and in particular those havingfrom 3 to 10 carbon atoms; more preferred are 3bromopropionic acid and4bromobutyric acid. The synthesis reaction between the functionalhydroxylic groups of HA (or the derivatives thereof) and a taxanecomponent such as paclitaxel, can be achieved by a process of indirector direct synthesis.

Indirect synthesis may lead to the formation of the following types ofcovalent bond between the spacer and HA or HA derivatives:

ester bond:

involving the carboxyl function of a suitably chosen spacer that isactivated by an activating agent such as, for example, a carbodiimide(Scheme 1 below);

involving the hydroxyl groups of HA or HA derivative that are brominatedor substituted with a tosyl group with subsequent nucleophilicsubstitution by the carboxyl of the suitably chosen spacer (Scheme 2below); or

involving the anhydride function of a suitably chosen spacer (Scheme 3below).

urethane or thiourethane bond:

involving the amino group of a suitably chosen spacer (Scheme 4 below);or

involving the isocyanate or isothiocyanate function of a suitably chosenspacer (Scheme 5 below).

ether bond:

involving the epoxy function of the (suitably chosen) spacer (Scheme 6below);

involving the hydroxyl groups of HA or HA derivative that are brominatedor substituted by a tosyl group, with subsequent nucleophilicsubstitution by the hydroxyl group of a suitably chosen spacer (Scheme 7below).

acetal or ketal bond:

involving the aldehyde and/or ketonic group of the suitably chosenspacer (Scheme 8 below);

involving the hydroxyl group of the suitably chosen spacer and requiringthe presence of a simple carbonyl compound, such as formaldehyde (Scheme9 below).

The above-described processes can be performed using agents activatingof the hydroxyl group of HA or HA derivatives, for example selected fromthe group consisting of carbonyldiimidazole anddi-(N-succimidyl)carbonate.

The direct synthesis reaction between the hydroxyl groups of HA or HAderivatives and a taxane such as paclitaxel, may lead to the formationof the following type of covalent bond: acetal bond:

involving the hydroxyl group of the taxane and the hydroxyl groups of HAor HA derivatives, which are covalently bounded by addition of a simplecarbonyl compound such as formaldehyde (Scheme 10)

The reaction between the carboxyl groups of HA or HA derivatives and ataxane such as paclitaxel, can be achieved by a process of direct orindirect synthesis.

Indirect synthesis may lead to the formation of the following types ofcovalent bond between the spacer and HA or HA derivatives:

ester bond:

the carboxylic group of the suitably chosen spacer, such as4-bromobutyric acid, is activated by an activating agent such as acarbodiimide and thus made suitable for synthesis with the hydroxylgroup of the taxane (preferably that on carbon at C2^(I)), such aspaclitaxel. Subsequently, by direct contact in an anhydrous solvent witha quaternary ammonium salt, in particular the tetrabutylammonium (TBA)salt of HA or HA derivative, a nucleophilic substitution is obtained ofthe carboxyl of HA or HA derivative to the bromine of the spacer. Inthis way an ester bond is formed between HA or HA derivative and thespacer, in turn bounded to paclitaxel. Alternatively, the nucleophilicsubstitution of the carboxyl group of HA or HA derivative to the bromineof the spacer may occur before the bond between the spacer itself andthe taxane (Scheme 11 below).

by using the activating agents of the carboxyl group of HA or HAderivative such as a carbodiimide, it is possible to obtain an esterbond between said group and the hydroxyl function of the (suitablychosen) spacer, previously or subsequently bound to paclitaxel (Scheme12 below).

amide bond:

activation of the carboxyl groups of HA or HA derivatives by anactivating agent, enables a linkage with the amino group of the suitablychosen spacer, with the exception of all the hydrazides, previously orsubsequently bound to a taxane such as paclitaxel (Scheme 13 below).

Direct synthesis can lead to the formation of the following types ofcovalent bond:

ester bond:

the activation of the carboxyl groups of HA or HA derivative by anactivating agent, enables its linkage with the hydroxyl group of thetaxane (Scheme 14 below);

activation of the hydroxyl of the taxane component by the activatingagent enables its linkage with the carboxylic function of HA or aderivative thereof (Scheme 14);

the following type of bond requires the bromide or tosylate of thetaxane. Said bond is prepared by nucleophilic substitution of thebromide or the tosyl group by the carboxyl group of HA or HA derivative(Scheme 15).

The synthesis reaction between the amino groups of deacetylated HA and ataxane component such as paclitaxel can come about by a process ofindirect or direct synthesis.

Indirect synthesis can lead to the formation of the following types ofcovalent bond between the spacer and HA:

amide bond:

involving the carboxylic group of a suitably chosen spacer (Scheme 16);

urethane or thiourethane bond:

involving the hydroxyl or thiolic group of a suitably chosen spacer(Scheme 17).

Direct synthesis can lead to the formation of the following type ofcovalent bond:

urethane bond:

involving the hydroxyl group of the taxane and the amino function ofdeacetylated HA (Scheme 18).

In the same way, the bond involving the spacer and a taxane such aspaclitaxel, may be of ester (Scheme 19), urethane or thiourethane(Scheme 20), acetal or ketal type (Scheme 21) and may require thepresence of an activating agent especially for the ester and urethanebonds.

The spacer can be bound to the taxane such as paclitaxel before or afterits linkage with the functional groups of HA or HA derivatives,depending on the type of functional groups of the suitably chosenspacer.

The percentage of direct or indirect linkage of the taxane, such aspaclitaxel, to HA or HA derivative may vary between 0.1% and 100% andpreferably between 0.1% and 35%.

The following examples are given to provide non-limiting illustrationsof the present invention.

EXAMPLE 1

Effect of the New Ester Derivative of HA with Paclitaxel in Nude MouseAfter Implant of Neoplastic Cells

For this experiment, we used human ovary adenocarcinoma cells, OVCAR-3cells, in immunodepressed nude mice belonging to the Athymic CD-1species.

Each mouse was inoculated by the intraperitoneal route with 5×10⁶ cancercells.

Experimental Design

Test drugs:

-   -   Taxol®, 5 animals treated    -   HYTAD1p20: ester derivative of HA covalently bound to paclitaxel        with 16% of esterification of the carboxyl (w/w). The molecular        weight of the HA used for synthesis of this new drug was 200,000        Da (see Example 7 for details of its preparation). Five animals        were used for this drug too.

Treated animals: 10 animals were first inoculated with OVCAR-3 cells.Five were used for the experiment with Taxol and another five for theexperiment with HYTAD1p20:

-   -   all ten animals subsequently received, by intraperitoneal        injection, 3 doses of pharmacological treatment (on the 6^(th),        13^(th) and 20^(th) days after inoculation of the cancer cells),        equal to 20 mg/kg body weight of Taxol® or 125 mg/kg body weight        of HYTAD (corresponding to 20 mg/mouse of paclitaxel).

Control animals: 5 animals were first inoculated with thecancer-inducing suspension of OVCAR-3 cells, after which they did notreceive any treatment.

Determination of the Survival Curve

The survival curve was calculated from the date of intervention to the92^(nd) day after inoculation of the cancer cells into the peritoneum.

Results: the results obtained are illustrated in FIG. 1.

Three control animals developed adenocarcinoma of the ovary and diedbetween the 70^(th) and 75^(th) days after inoculation of the cancercells.

On the 92^(nd) day after intervention, the last day of the experiment,none of the animals that had received pharmacological treatment withpaclitaxel or HYTAD had died.

EXAMPLE 2

In vitro Experiment

The aim of the in vitro experiment was mainly to define the activityprofile of the new ester derivatives of HA bound to paclitaxel and toassess/compare the antineoplastic activity of the HYTAD derivatives vspaclitaxel, thus determining their pharmacological potential compared tothe antineoplastic drug.

Experimental Design:

Test products:

-   -   Taxol®: reference product    -   HYTAD1p20—HYTAD2p20—HYTAD2p10: ester derivatives of HA        covalently bound to paclitaxel with 16% of esterification of the        carboxyl (w/w) (in the case of HYTAD1p20, the molecular weight        of the HA used in the synthesis of this new drug is 200,000 Da)        (see Example 7 for details of its preparation) or 22% (in the        case of HYTAD2p20, the molecular weight of the HA used is 39,000        Da), or 6.8% (in the case of HYTAD2p10, the molecular weight of        the HA used is 39,000 Da).

Cell Lines

Cell Lines of Human Origin

Four cell lines of human breast cancer were used. All four of the testcell strains normally respond to paclitaxel and express the receptorCD44 apparently with the same amplification.

-   -   MCF-7    -   MDA-MB-231    -   MDA-MB-468    -   SKBR-3

Experimental Protocol:

1) the test cell line is plated at a concentration of 3,000 cells perwell, on a flat-bottomed, 96-well plate;

2) 24 hours later, the cells are supplemented with the test solutionssuitably diluted in culture medium;

3) after another 72 hours, the cells are tested by colorimetry with3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT);by assessing cell viability, this test also reveals the differentsensitivity of the cells to the test drug. This is possible becausemitochondrial dehydrogenase is able to reduce the tetrazolium salts(yellow) into blue formazan crystals. The greater or lesser intensity ofcolour is assessed by spectrophotometry (Dezinot, F. et al., J. Immunol.Methods, 1986, 22 (89): 271-277).

Results

Hereafter we report, in table and graph form in FIG. 2, the resultsobtained in terms of IC₅₀ (the concentration of drug necessary toinhibit cell growth by 50% with regard to the test product and thedifferent cell lines used).

In FIG. 2, the axis of abscissas represents the pharmacological power,expressed as IC₅₀ and calculated as the ratio between the molarconcentrations, vs the reference product (paclitaxel) which isconventionally taken to have a value of nil. Consequently, the dashesindicate a pharmacological power that is greater than the referenceproduct.

IC₅₀ (expressed as nM or μM of paclitaxel or its HYTAD derivatives inthe culture medium) Cell lines Taxol ® HYTAD2p20 HYTAD1p20 HYTAD2p10Breast cancer cell lines MCF/7  3.5 nM 0.86 nM 0.024 nM 0.68 nM MDA/0.35 nM 2.58 nM — 0.24 μM MB/231 MDA/  9.4 nM —  0.18 nM — MB/468 SKBR/30.23 nM — — 0.14 nM

Conclusions

As reported in the literature, all the cell lines used are sensitive totaxol, a drug mainly used to treat metastatic carcinoma of the breastand of the ovary. As regards the breast cancer cell lines, the variousHYTAD proved to be considerably stronger than paclitaxel, with a factorof +150 with regard to HYTAD1p20 on cancer cell line MCF-7.

EXAMPLE 3

Effect of ACP® Gel in Nude Mice After Implantation of Neoplastic Cells.

For this experiment, we used human colic carcinoma HT29 cells Inimmunodepressed nude mice belonging to the Athymic Nude-nu (nu/nu)species. Each animal was anaesthetised and 0.3 ml of an HT29 cellsuspension was injected into its peritoneal cavity at a concentration of166,000 cells/ml. Thus, each mouse received 50,000 cancer cells.

Experimental Design:

Treated animals: 113 animals were first inoculated with HT29 andimmediately afterwards they received a single dose of treatment equal to0.2 ml of ACP gel 40 mg/ml;

Control animals: 117 animals were inoculated with HT29 cancer cellsuspension but received no treatment.

Survival curve: the survival curve was calculated from the day ofinoculation up to the day of death. Deaths were either ascertained orcaused by the sacrifice of animals whose weight had dropped by more than20% of their starting weight, and in the case of hemoperitoneumindicating diffuse metastases. The percentage of survival in the twogroups was determined daily and expressed as a graph to obtain the curvereported in FIG. 3.

The experiment lasted 120 days, after which all the surviving animalswere sacrificed and examined necroscopically to check for the presenceof abdominal tumours.

Results: 32 animals out of 230 had not developed any notable neoplasia.22 of these animals belonged to the group of mice treated with ACP® gel,10 to the control group.

ACP® gel: 19.5% of the treated animals did not develop neoplasia;

Control: 8.5% of the control animals did not develop neoplasia.

EXAMPLE 4

Preparation of HA with a Molecular Weight of Between 5,000 and 10,000Daltons (for Possible Synthesis of HA-Paclitaxel withLow-Molecular-Weight HA)

2.40 g of sodium HA with a molecular weight of 990,000 Da is dissolvedin 240 ml of a solution of 0.15M NaCl. This is then supplemented with7.9 ml of a 14% solution of NaOCl. At a constant temperature of +4° C.,the solution is sonicated for 120 minutes at a frequency of 20 Hz and at150 W. Once the reaction is complete, the pH is adjusted to 6.5 with0.1N HCl and the solution is then precipitated in 1.000 ml of a 2:1mixture of methanol-acetone. The product is collected by filtration andvacuum-dried for 48 hours at 45° C. 1.65 g of sodium salt is thusobtained. High pressure liquid chromatography (HPLC)-GPC analysisreveals that the fraction of HA obtained has a mean molecular weight(MW) of 5,850, a mean numerical molecular weight (MN) of 3,640 and apolydispersity index of 1.61.

EXAMPLE 5

Preparation of an Ester Derivative of HA Bound to Paclitaxel WithEsterification of the Carboxyl of about 4% w/w

51 mg of paclitaxel is dissolved in CH₂Cl₂ and the solution issupplemented with 104 mg of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 20 mg of4-bromobutyric acid. Subsequently, the solution is partitioned in water.After eliminating the carbodiimide and bromide residues, the reactionsolvent is dried with anhydrous sodium sulfate and eliminated with arotary evaporator. 21 mg of product thus obtained is dissolved inn-methyl-pyrrolidone (NMP) and added to a 20 mg/ml solution of HAsalified with tetrabutylammonium. (TBA) in NMP (200 mg in 10 ml NMP).After seven days' reaction at ambient temperature, the solution isdiluted with 5 ml of water and 1 ml of saturated NaCl solution. Thesolution thus obtained is stirred for 1 hour to enable the exchange ofsodium with the TBA ion.

Subsequently, ethanol is slowly added a drop at a time and thefilamentous product thus obtained is dissolved in water, dialysed andlastly freeze-dried.

EXAMPLE 6

Preparation of an Ester Derivative of HA With Paclitaxel WithEsterification at the Carboxyl of about 10% w/w

As in Example 5, 308.7 mg of paclitaxel dissolved in 15 ml ofdichloromethane is supplemented with 117.2 mg of 4-bromobutyric acid and614.1 mg of EDC. Subsequently, water is added to the solution toeliminate all the bromide and carbodiimide. The organic solution thusobtained is supplemented with sodium sulphate to dehydrate it while thesolvent is eliminated with a rotary evaporator. Finally, 363 mg ofintermediate product is obtained.

175 mg of intermediate product thus obtained is added to 1 g of HA-TBAdissolved in anhydrous NMP. The solution is stirred at ambienttemperature for 7 days, after which 20 ml of water and 4 ml of asaturated NaCl solution are added. It is stirred for 1 hour to enablethe exchange of sodium with the TBA ion. Subsequently ethanol is slowlyadded a drop at a time and the filamentous product this obtained isdissolved in water, dialysed and lastly freeze-dried.

EXAMPLE 7

Preparation of an Ester Derivative of HA With Paclitaxel WithEsterification at the Carboxyl of about 16% w/w

164 mg of intermediate product, obtained according to the proceduredescribed in the previous examples No. 5 and 6, is added to a solutionof 680 mg of HA-TBA dissolved in 25 ml of anhydrous NMP. After 7 days'reaction at ambient temperature, the solution is supplemented with 20 mlof water and 4 ml of saturated NaCl solution. After 1 hour, ethanol isslowly added a drop at a time. The product obtained is collected byfiltration and dissolved in water, dialysed and, when the conductibilityof the dialysis solution has dropped below 10 μS, it is frozen. Thefrozen solution is then freeze-dried.

EXAMPLE 8

Preparation of an Ester Derivative of HA With Paclitaxel WithEsterification at the Hydroxyl of about 10% w/w

102.6 mg of paclitaxel is dissolved in 5 ml of dichloromethane and thesolution is supplemented with 20.4 mg of succinic anhydride. Three hourslater, the solvent is eliminated by evaporation using a rotaryevaporator. The product thus obtained is dissolved in 5 ml of dimethylsulphoxide (DMSO) with low water content, and 27.3 mg ofdicyclo-hexyl-carbodiimide is added. About 5 minutes later, the solutionis supplemented with a solution of HA-TBA, obtained by dissolving 327 mgof polymer in 15 ml of DMSO with low water content. The solution isstirred at ambient temperature for about 24 hours. Subsequently, a fewml of water and 3 ml of a saturated NaCl solution are added to thesolution. After 1 hour it is precipitated by adding ethanol. Thefilamentous product collected by filtration is dissolved in water,dialysed and lastly freeze-dried.

EXAMPLE 9

Preparation of an Ester Derivative of HA With Paclitaxel WithEsterification at the Carboxyl of about 4% w/w

510.1 mg of paclitaxel dissolved in 6 ml of dichloromethane issupplemented with 95.4 mg of 3-3 bromopropionic acid and 525.0 mg ofEDC. Subsequently, water is added to the solution to eliminate thebromide and the carbodiimide by partitioning, while 10 volumes of waterare used to eliminate the reagents. The organic solution is supplementedwith sodium sulphate to dehydrate it and the solvent is eliminated witha rotary evaporator.

155.5 mg of intermediate product thus obtained is added to 1.46 g ofHA-TBA dissolved in anhydrous NMP and the solution thus obtained isstirred at ambient temperature for 7 days. Subsequently, 20 ml of waterand 4 ml of saturated NaCl solution are added. The solution is stirredfor 1 hour to enable the exchange of sodium with the TBA ion. Thenethanol is slowly added a drop at a time and the filamentous productthus obtained is dissolved in water, dialysed and lastly freeze-dried.

EXAMPLE 10

Preparation of an Ester Derivative of Hyaluronic Acid WithEsterification at the Carboxyl of about 30% w/w

500 mg of paclitaxel is dissolved in CH₂Cl₂ and the solution issupplemented with 397.6 mg of13-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 300.9 mg of4-bromobutyric acid. Subsequently, the solution is partitioned in water.Once the carbodiimide and bromide residues have been eliminated, thereaction solvent is dried with anhydrous sodium sulphate and eliminatedwith a rotary evaporator.

The product thus obtained is dissolved in NMP and added to a solutioncontaining ˜20 mg/ml of hyaluronic acid salified with TBA in NMP (1.95 gin 100 ml NMP).

After 7 days' reaction at ambient temperature, the solution is dilutedwith 20 ml of water and 4.5 ml of a saturated NaCl solution. Thesolution is stirred for 1 hour to enable the exchange of sodium with theTBA ion. Subsequently, ethanol is slowly added a drop at a time and thefilamentous product thus obtained is dissolved in water and dialysed andlastly freeze-dried.

EXAMPLE 11

Preparation of the Partial Autocrosslinked Ester (about 10%Substitution) of HA With 8% Paclitaxel w/w

3.10 g of HA salified with TBA is solubilised in 150 ml of DMSO with alow water content at ambient temperature. The solution is thensupplemented with 541.0 mg of intermediate paclitaxel obtained accordingto the method described in examples 5, 6 and 7. Once it has been left toreact for 7 days at ambient temperature, the reaction solution issupplemented with 126.5 g of triethylamine and the whole is stirred for30 minutes.

A solution of 319.5 g of 2-chloro-1-methyl-pyridine iodide in 30 ml ofDMSO is slowly added a drop at a time over a 45-minute interval and themixture is left at 30° C. for 15 hours.

A solution formed by 50 ml of water and 1.7 g of sodium chloride isadded and the resulting mixture is poured slowly into 400 ml of acetonewhile stirring continuously. A precipitate is formed that is filteredand washed three times with 50 ml of acetone water 5:1 and three timeswith acetone (50 ml). The final product thus obtained is vacuum-dried at38° C.

EXAMPLE 12

Tests of the Solubility of the HA-Paclitaxel Ester Obtained According toExample 5 in a 5% Glucose Solution.

14.6 mg of an HA-paclitaxel product obtained by esterification accordingto Example 7 (starting from HA with a molecular weight of 200 kDa) witha degree of substitution at the carboxyl of 16.3% w/w, was dissolved in1 ml of an aqueous solution of 5% glucose. The solution, stirred with amagnetic stirring bar, can be filtered through a 0.20 μm sterilityfilter fitted on a syringe. The concentration of paclitaxel in thesolution is 2.38 mg/ml.

We also attempted to find the maximum concentration of product per ml of5% glucose aqueous solution. At a concentration of 32.8 mg ofHA-paclitaxel product per ml of glucose solution, a viscous solution isobtained with a concentration of paclitaxel of 5.35 mg/ml.

EXAMPLE 13

Tests to Recover Paclitaxel from Human Plasma

A solution is prepared that is constituted by 101.3 mg of HA-paclitaxelIn 10 ml of water. The HA-paclitaxel is prepared as described in Example7.

The recovery test is performed by placing 40 mg of the above describedsolution in contact with 2 ml of human plasma at 37° C.

To determine the paclitaxel that is released into the plasma bydetaching itself from the HA, three contact times were set: 6, 30 and 60minutes. At the end of each contact interval, the paclitaxel wasextracted from the plasma-HA-paclitaxel solution with 3 rinses, eachwith 1.5 ml of terbutylmethylether (TBME), which were collectedtogether, evaporated to dryness by natural evaporation at 65° C., andresuspended in 400 μl of absolute ethanol to determine the content ofthe drug in question by HPLC (high pressure liquid chromatography). Theresults obtained are shown in FIG. 4: after 6 minutes more than 80% ofthe paclitaxel had become detached from the HA and the percentage hadnot increased by the later observation times.

The invention being thus described, it is clear that these methods canbe modified in various ways. Such modifications are not to be consideredas divergences from the spirit and purpose of the invention, and anysuch modification that may appear evident to an expert in the fieldcomes within the scope of the following claims.

1. A taxane covalently bonded to hyaluronic acid or to a hyaluronic acidderivative, wherein the covalent bond is formed between hydroxyl groupsof the taxane and carboxyl groups or hydroxyl groups of hyaluronic acidor of hyaluronic acid derivatives, or amino groups of deacetylatedhyaluronic acid, optionally by means of a spacer linking the taxane tohyaluronic acid or hyaluronic acid derivative, with the proviso that thesaid spacer is different from a hydrazide.
 2. The taxane according toclaim 1, wherein the taxane is selected from between paclitaxel anddocetaxel.
 3. The taxane according to claim 1, wherein the said taxaneis paclitaxel.
 4. The taxane according to claim 1, wherein thehyaluronic acid has a molecular weight of between 400 and 3×10⁶ Daltons.5. The taxane according to claim 4, wherein the hyaluronic acid has amolecular weight of between 400 and 1×10⁶ Daltons.
 6. The taxaneaccording to claim 4, wherein the hyaluronic acid has a molecular weightof between 400 and 230,000 Daltons.
 7. The taxane according to claim 1,wherein the hyaluronic acid is salified with organic and/or inorganicbases.
 8. The taxane according to claim 1, wherein the hyaluronic acidderivative is selected from the group consisting of esters of hyaluronicacid with alcohols of the aliphatic, araliphatic, cycloaliphatic,aromatic, cyclic and heterocyclic series, said esters having anesterification degree equal to or lower than 50%.
 9. The taxaneaccording to claim 1, wherein the hyaluronic acid derivative is selectedfrom the group consisting of amides of hyaluronic acid with amines ofthe aliphatic, araliphatic, cycloaliphatic, aromatic, cyclic andheterocyclic series, said amides having an amidation degree of between0.1% and 10%.
 10. The taxane according to claim 1, wherein thehyaluronic acid derivative is selected from the group consisting ofO-sulphated derivatives of hyaluronic acid up to the 4^(th) degree ofsulphation.
 11. The taxane according to claim 1, wherein the hyaluronicacid derivative is selected from the group consisting of inner esters ofhyaluronic acid having an esterification degree equal to or lower than15%.
 12. The taxane according to claim 1, wherein the hyaluronic acidderivative is selected from the group consisting of deacetylates ofhyaluronic acid, coming from deacetylation of the N-acetyl-glucosamineunit and having a deacetylation degree of between 0.1% and 30%.
 13. Thetaxane according to claim 1, wherein the hyaluronic acid derivative isselected from the group consisting of percarboxylated derivatives ofhyaluronic acid obtained from the oxidation of the primary hydroxyl ofthe N-acetyl-glucosamine unit, having a percarboxylation degree ofbetween 1 and 100%.
 14. The taxane according to claim 1, wherein thecovalent bond is formed between hydroxyl groups of the taxane andhydroxyl groups of hyaluronic acid or of hyaluronic acid derivative. 15.The taxane according to claim 1, wherein the covalent bond is formedbetween hydroxyl groups of the taxane and carboxyl groups of hyaluronicacid or of hyaluronic acid derivative.
 16. The taxane according to claim1, wherein the covalent bond is formed between hydroxyl groups of thetaxane and amino groups of deacetylated hyaluronic acid.
 17. The taxaneaccording to claim 1, wherein the spacer linking the taxane tohyaluronic acid or hyaluronic acid derivative, is selected from thegroup consisting of aliphatic or araliphatic chains, linear or branched,substituted with one or more groups chosen from hydroxyl, carboxyl,carbonyl, epoxide, acylchloride, thiol, nitryl, halogen, anhydride,isocyanate, isothiocyanate and amino groups.
 18. The taxane according toclaim 17, wherein the spacer is selected from the group consisting ofcarboxylic acids having from 2 to 18 carbon atoms in the aliphatic oraraliphatic chain, substituted with bromine.
 19. The taxane according toclaim 17, wherein the spacer is selected from the group consisting ofcarboxylic acids having from 3 to 10 carbon atoms in the aliphatic oraraliphatic chain, substituted with bromine.
 20. The taxane according toclaim 17, wherein the spacer is selected from between 3-bromopropionicacid and 4-bromobutyric acid.
 21. The taxane according to claim 1,wherein the covalent bond is an ester bond between the spacer and thehydroxyl groups of hyaluronic acid or of hyaluronic acid derivative. 22.The taxane according to claim 1, wherein the covalent bond is a urethaneor thiourethane bond between the spacer and the hydroxyl groups ofhyaluronic acid or of hyaluronic acid derivative.
 23. The taxaneaccording to claim 1, wherein the covalent bond is an ether bond betweenthe spacer and the hydroxyl groups of hyaluronic acid or of hyaluronicacid derivative.
 24. The taxane according to claim 1, wherein thecovalent bond is an acetal or ketal bond between the spacer and thehydroxyl groups of hyaluronic acid or of hyaluronic acid derivative. 25.The taxane according to claim 1, wherein the covalent bond is an acetalbond between the hydroxyl groups of hyaluronic acid or of hyaluronicacid derivative and the taxane.
 26. The taxane according to claim 1,wherein the covalent bond is an ester bond between the spacer and thecarboxyl groups of hyaluronic acid or of hyaluronic acid derivative. 27.The taxane according to claim 1, wherein the covalent bond is an amidebond between the spacer and the carboxyl groups of hyaluronic acid or ofhyaluronic acid derivative.
 28. The taxane according to claim 1, whereinthe covalent bond is an ester bond between the carboxyl groups ofhyaluronic acid or of hyaluronic acid derivative and hydroxyl groups ofthe taxane.
 29. The taxane according to claim 1, wherein the covalentbond is an amide bond between the spacer and the amino groups ofdeacetylated hyaluronic acid.
 30. The taxane according to claim 1,wherein the covalent bond is a urethane or thiourethane bond between thespacer and the amino groups of deacetylated hyaluronic acid.
 31. Thetaxane according to claim 1, wherein the covalent bond is a urethanebond between the amino groups of deacetylated hyaluronic acid andhydroxyl groups of the taxane.
 32. The taxane according to claim 8,wherein the hyaluronic acid is esterified after the formation of thecovalent bond with the taxane.
 33. The taxane according to claim 11,wherein the hyaluronic acid is esterified after the formation of thecovalent bond with the taxane.
 34. The taxane according to claim 1,wherein the covalent bond is an ester bond between the taxane and thespacer.
 35. The taxane according to claim 1, wherein the covalent bondis a urethane or thiourethane bond between the taxane and the spacer.36. The taxane according to claim 1, wherein the covalent bond is anacetal or ketal bond between the taxane and the spacer.
 37. The taxaneaccording to claim 1, wherein the bond percentage between hyaluronicacid and the taxane is between 0.1% and 100%.
 38. The taxane accordingto claim 37, wherein the bond percentage between hyaluronic acid and thetaxane is between 0.1% and 35%.
 39. The taxane according to claim 1,wherein the hyaluronic acid or hyaluronic acid derivative enhances theanticancer action of the taxane.
 40. The taxane according to claim 11,wherein the inner ester of hyaluronic acid enhances the anticanceraction of taxane.
 41. The taxane according to claim 26, wherein thehyaluronic acid enhances the anticancer action of taxane.
 42. Apharmaceutical composition comprising as the active substance at least ataxane covalently bonded to hyaluronic acid or to a hyaluronic acidderivative as defined in claim 1, in combination with pharmaceuticallyacceptable excipients and diluents.
 43. The pharmaceutical compositionaccording to claim 42, for administration by the oral, intravenous,arterial, intramuscular, subcutaneous, intraperitoneal or transdermalroute, or by direct injection into a tumour site.
 44. The pharmaceuticalcomposition according to claim 42, for administration by the oral route.45. The pharmaceutical composition according to claim 42, wherein thehyaluronic acid or the hyaluronic acid derivative is able to release thetaxane into the administration site.
 46. The pharmaceutical compositionaccording to claim 42, further comprising one or more biologically orpharmacologically active substances.
 47. The pharmaceutical compositionaccording to claim 46, wherein the said biologically orpharmacologically active substances are selected from the groupconsisting of steroids, hormones, trophic factors, proteins, vitamins,non-steroid anti-inflammatory drugs, chemotherapy drugs, calciumblockers, antibiotics, antivirals, interleukines and cytokines.
 48. Thepharmaceutical composition according to claim 46, wherein the saidbiologically or pharmacologically active substance is interferon. 49-54.(canceled)
 55. Stents and medical devices coated by a taxane covalentlybonded to hyaluronic acid or to a hyaluronic acid derivative as definedin claims
 144. 56. A process for the preparation of a taxane covalentlybonded to hyaluronic acid or to a hyaluronic acid derivative wherein thecovalent bond is an ester bond, said process comprising the followingsteps: A) activating the hydroxyl group of the taxane or, respectively,the carboxyl group of hyaluronic acid or hyaluronic acid derivative bymeans of an activating agent; B) adding the hyaluronic acid orhyaluronic acid derivative or, respectively, the taxane dissolved in asuitable solvent; C) optionally purifying the so obtained product.
 57. Aprocess for the preparation of a taxane covalently bonded to hyaluronicacid or to a hyaluronic acid derivative wherein the covalent bond is anester bond, said process comprising the following steps: A′) preparingthe bromide or tosylate of the taxane; B′) carrying out the nucleophilicsubstitution of the bromide or tosylate of taxane coming from step A′)by the carboxyl group of hyaluronic acid or of hyaluronic acidderivative; C′) optionally purifying the product obtained.
 58. A processfor the preparation of a taxane covalently bonded to deacetylatedhyaluronic acid wherein the covalent bond is a urethane or thiourethanebond, said process comprising the following steps: D) activating thehydroxyl group of taxane by means of an activating agent; E) addingdeacetylated hyaluronic acid dissolved in a suitable solvent; F)optionally purifying the so obtained product.
 59. A process for thepreparation of a taxane covalently bonded to hyaluronic acid orhyaluronic acid derivative wherein the covalent bond is an acetyl bond,said process comprising the following steps: G) preparing a solutioncontaining hyaluronic acid or hyaluronic acid derivative and the taxanein a suitable solvent; H) adding a simple carbonyl compound such asformaldehyde; I) optionally purifying the so obtained product.
 60. Aprocess for the preparation of a taxane covalently bonded to hyaluronicacid or hyaluronic acid derivative by means of a spacer having at leasta carboxyl group and linking the hydroxyl group hyaluronic acid or thehyaluronic acid derivative by an ester bond, said process comprising thefollowing steps: L) activating the carboxyl group of the spacer,possibly previously bounded to the taxane; M) adding hyaluronic acid orhyaluronic acid derivative; N) optionally purifying the so obtainedproduct, and reacting with the taxane if not previously bounded to thespacer.
 61. A process for the preparation of a taxane covalently bondedto hyaluronic acid or hyaluronic acid derivative by means of a spacerhaving at least a carboxyl group and linking the hydroxyl grouphyaluronic acid or the hyaluronic acid derivative by an ester bond, saidprocess comprising the following steps: L′) substituting the hydroxylgroup of hyaluronic acid or hyaluronic acid derivative with a tosylgroup or bromide; M′) adding the spacer, possibly previously bounded tothe taxane; N′) optionally purifying the so obtained product, andreacting with the taxane if not previously bounded to the spacer.
 62. Aprocess for the preparation of a taxane covalently bonded to hyaluronicacid or hyaluronic acid derivative by means of a spacer having at leastan anhydride group and linking the hydroxyl group hyaluronic acid or thehyaluronic acid derivative by an ester bond, said process comprising thefollowing steps: L″) adding the spacer to a solution containinghyaluronic acid or hyaluronic acid derivative; M″) optionally purifyingthe so obtained product; N″) reacting the product coming from step L″)or M″) with the taxane.
 63. A process for the preparation of a taxanecovalently bonded to hyaluronic acid or hyaluronic acid derivative bymeans of a spacer having at least an amino group and linking thehydroxyl group hyaluronic acid or the hyaluronic acid derivative by aurethane or thiourethane bond, said process comprising the followingsteps: O) activating the hydroxyl group of hyaluronic acid or ofhyaluronic acid derivative by means of an activating agent; P) addingthe spacer, possibly previously bounded to the taxane; Q) optionallypurifying the so obtained product, and reacting with the taxane if notpreviously bounded to the spacer.
 64. A process for the preparation of ataxane covalently bonded to hyaluronic acid or hyaluronic acidderivative by means of a spacer having at least an isocyanate orisothiocyanate group and linking the hydroxyl group hyaluronic acid orthe hyaluronic acid derivative by a urethane or thiourethane bond, saidprocess comprising the following steps: O′) adding hyaluronic acid orhyaluronic acid derivative to a solution comprising the spacer, possiblypreviously bonded to the taxane; P′) optionally purifying the soobtained product, and reacting with the taxane if not previously bondedto the spacer.
 65. A process for the preparation of a taxane covalentlybonded to hyaluronic acid or hyaluronic acid derivative by means of aspacer having at least an epoxy group and linking the hydroxyl grouphyaluronic acid or the hyaluronic acid derivative by an ether bond, saidprocess comprising the following steps: R) adding the spacer possiblypreviously bounded to the taxane, to a solution of hyaluronic acid orhyaluronic acid derivative, in the presence of an acid or basiccatalyst; S) optionally purifying the so obtained product, and reactingwith the taxane if not previously bounded to the spacer.
 66. A processfor the preparation of a taxane covalently bonded to hyaluronic acid orhyaluronic acid derivative by means of a spacer having at least anhydroxyl group and linking the hydroxyl group hyaluronic acid or thehyaluronic acid derivative by an ether bond, said process comprising thefollowing steps: R′) substituting the hydroxyl group of hyaluronic acidor hyaluronic acid derivative with a tosyl group or bromide; S′) addingthe spacer to the product coming from step R′) in a basic environment;T′) optionally purifying the so obtained product; U′) reacting theproduct coming from step S′) or T′) with the taxane.
 67. A process forthe preparation of a taxane covalently bonded to hyaluronic acid orhyaluronic acid derivative by means of a spacer having at least acarbonyl group and linking the hydroxyl group hyaluronic acid or thehyaluronic acid derivative by an acetal or ketal bond, said processcomprising the following steps: V) adding the spacer to a solutioncontaining hyaluronic acid or hyaluronic acid derivative in acid orbasic environment; W) optionally purifying the so obtained product; Z)reacting the product coming from step V) or W) with the taxane.
 68. Aprocess for the preparation of a taxane covalently bonded to hyaluronicacid or hyaluronic acid derivative by means of a spacer having at leastan hydroxyl group and linking the hydroxyl group hyaluronic acid or thehyaluronic acid derivative by an acetal or ketal bond, said processcomprising the following steps: V′) adding a simple carbonyl compound,such as formaldehyde, to a solution containing hyaluronic acid orhyaluronic acid derivative and a spacer possibly previously bonded tothe taxane; W′) optionally purifying the so obtained product, andreacting with the taxane if not previously bonded to the spacer.
 69. Aprocess for the preparation of a taxane covalently bonded to hyaluronicacid or hyaluronic acid derivative by means of a spacer having at leastan hydroxyl group and linking the carboxyl group of hyaluronic acid orthe hyaluronic acid derivative by an ester bond, said process comprisingthe following steps: a) adding an activating agent to a solutioncontaining hyaluronic acid or hyaluronic acid derivative; b) adding thespacer possibly previously bound to the taxane, to the solution comingfrom step a); c) optionally purifying the so obtained product, andreacting with the taxane if not previously bonded to the spacer.
 70. Aprocess for the preparation of a taxane covalently bonded to hyaluronicacid or hyaluronic acid derivative by means of a spacer having at leastan halogen, such as bromine, and linking the carboxyl group ofhyaluronic acid or the hyaluronic acid derivative by an ester bond, saidprocess comprising the following steps: a′) adding the spacer possiblypreviously bounded to the taxane, to a solution of hyaluronic acid orhyaluronic acid derivative; b′) optionally purifying the so obtainedproduct, and reacting with the taxane if not previously bounded to thespacer.
 71. A process for the preparation of a taxane covalently bondedto hyaluronic acid or hyaluronic acid derivative by means of a spacerhaving at least an amino group and linking the carboxyl group ofhyaluronic acid or the hyaluronic acid derivative by an amide bond, saidprocess comprising the following steps: d) adding an activating agent toa solution of hyaluronic acid or hyaluronic acid derivative; e) addingthe spacer possibly previously bonded to the taxane to the solutioncoming from step d); f) optionally purifying the so obtained product,and reacting with the taxane if not previously bounded to the spacer.72. A process for the preparation of a taxane covalently bonded todeacetylated hyaluronic acid by means of a spacer having at least acarboxyl group and linking the amino group of deacetylated hyaluronicacid by an amide bond, said process comprising the following steps: g)activating with an activating agent the carboxyl group of the spacerpossibly previously bounded to the taxane; h) adding a solutioncontaining deacetylated hyaluronic acid; i) optionally purifying the soobtained product, and reacting with the taxane if not previously boundedto the spacer.
 73. A process for the preparation of a taxane covalentlybonded to deacetylated hyaluronic acid by means of a spacer having atleast an hydroxyl group and linking the amino group of deacetylatedhyaluronic acid by a urethane or thiourethane bond, said processcomprising the following steps: l) activating with an activating agentthe hydroxyl group of the spacer possibly previously bonded to thetaxane; m) adding the solution containing deacetylated hyaluronic acid;n) optionally purifying the so obtained product, and reacting with thetaxane if not previously bonded to the spacer.
 74. A therapeutic methodfor the treatment of tumours, which comprises administering to a subjectin a need for such a treatment a therapeutically effective amount oftaxane covalently bonded to hyaluronic acid or to a hyaluronic acidderivative as defined in claim
 1. 75. The therapeutic method accordingto claim 74, wherein the treatment of tumours comprises chemotherapy forbreast cancer, cancer of the ovary and/or endometrium, melanoma, lungcancer, cancer of the liver, of the prostate and/or bladder, gastricand/or intestinal cancer, leukaemia and Kaposi's sarcoma.
 76. Atherapeutic method for the treatment of auto-immune pathologies, whichcomprises administering to a subject in a need for such a treatment atherapeutically effective amount of taxane covalently bonded tohyaluronic acid or to a hyaluronic acid derivative as defined inclaim
 1. 77. The therapeutic method according to claim 76, wherein thesaid auto-immune pathologies are selected from the group consisting ofrheumatoid arthritis, Hashimoto's thyroiditis, systemic lupuserythematosus, and auto-immune glomerulonephritis.
 78. A therapeuticmethod for the treatment of restenosis, which comprises administering toa subject in a need for such a treatment a therapeutically effectiveamount of taxane covalently bonded to hyaluronic acid or to a hyaluronicacid derivative as defined in claim
 1. 79. A process for the manufactureof stents and medical devices, comprising the step of coating saidstents and medical devices by taxane covalently bonded to hyaluronicacid or to a hyaluronic acid derivative as defined in claim 1.